RNA polymerase (RNAP) transcribes RNA messages from DNA templates in all living cells. This is a central step in gene expression that is highly regulated in all organisms, from bacteria to humans. Dysregulation of transcription plays a large role in numerous disease states, such as cancer. It has recently been suggested that the process of transcription is not only regulated at the level of initiation (recruitment of RNAP to te promoter), but also by RNAP pausing at specific locations during elongation. The biological consequences of regulated transcription pausing are not well understood, hindering our ability to understand the relationship between transcription pausing and disease. The most well studied system with respect to transcription pausing mechanisms is the bacterium E. coli. The focus of this proposal is to investigate the biological consequences of transcription pausing by examining how it impacts the folding of RNA as it is revealed during transcription. Certain transcripts in E. coli contain structured RNA regions, termed riboswitches, that fold into competing structures based on the presence or absence of a small-molecule metabolite, and directly regulate gene expression in the cell. Riboswitches are thought to operate under kinetic control in the cell, making co-transcriptional RNA folding an essential determinant of their ability to recognize a metabolite and alter gene expression. Riboswitches have been proposed as novel antibacterial drug targets, and can additionally be considered models for eukaryotic splice-site structures, the folding and function of which also occur co-transcriptionally. This project will utilize bacterial riboswitches as a model system to explore the hypothesis that transcription pausing is required for co-transcriptional folding and function of cellular RNAs in vivo. The aims of this proposal are to (i) determine whether pausing of RNA polymerase regulates riboswitch function in vivo and identify which cellular factors are required for this regulation and (ii) elucidate the specific mechanistic consequences of transcription pausing for the folding of riboswitches. The use of E. coli as a model system will allow the use of sophisticated genetic tools to determine the effects of perturbing transcription pausing on riboswitch function and will facilitate a genetic screen to reveal as-of-yet undiscovered interactions that are required for riboswitch function in vivo, eithe between transcription factors and RNAP, or even between transcription factors and the RNA. In vivo studies will be complemented by detailed biochemical investigations to dissect the mechanistic basis for observed effects of transcription pausing on riboswitch RNA folding and function. In addition to laying a foundation for understanding how dysregulated transcription pausing can contribute to disease, this project will facilitate efforts to target riboswitches for antibacterial drugs, by significantly increasing our understanding of riboswitch function in vivo. PUBLIC HEALTH RELEVANCE: The dysregulation of transcription is common in many diseases, such as cancer, but the importance of transcription pausing as a regulatory mechanism is relatively unexplored. The work proposed here will lay a foundation for understanding the biological function of transcription elongation regulation by investigating the hypothesis that transcription pausing controls the folding and function of essential RNA structures in living cells. Additionally, this work will enhance our understanding of the in vivo mechanisms of bacterial riboswitches (the model system), which will help to inform the use of riboswitches as antibacterial drug targets.